The broad objective of this project is to understand how bacteria, as either pathogens or mutualists, sense and respond to host organisms. Members of the alphaproteobacteria class, which includes Agrobacterium (an extracellular plant pathogen), Brucella (an intracellular human and animal pathogen) and Sinorhizobium (an intracellular plant symbiont), need to secrete cyclic glucans in order to invade their hosts. Using the plant symbiont Sinorhizobium meliloti as a model for chronic infection, a novel `two-component'phosphotransfer system required for cyclic glucan production and host invasion has been discovered. This signaling module is encoded on a three-gene operon (feuN-feuP-feuQ) on the S. meliloti chromosome. The response regulator component, FeuP, activates transcription of the cyclic glucan exporter gene ndvA. The sensor kinase component, FeuQ, is also required for ndvA transcriptional activation and responds to external osmotic conditions. feuN encodes a small conserved protein of unknown function, and unlike feuP and feuQ, feuN is essential for cell viability. However, this lethality is suppressed if feuQ is deleted, suggesting a functional interaction between FeuN and the FeuP/Q two-component system. Recent experiments suggest that FeuN is a negative regulator of signaling through the FeuP/Q pathway. Two central questions emerge from these findings: 1) What is the mechanism by which the novel protein FeuN influences signaling through the FeuP/Q pathway? and 2) What is the specific cause of lethality in a feuN deletion mutant? In the first aim of this project, the subcellular localization and topology of FeuN will be determined, using marker-enzyme and cellular fractionation approaches. In the second aim, the functional interaction between FeuN and FeuP or FeuQ will be characterized using genetic and biochemical methods. Finally the third aim examines the specific cause of lethality in feuN mutant cells, by analyzing suppressor mutants, and ectopically expressing known gene targets of the FeuP/Q pathway. This investigation will provide important insights on a novel mechanism of gene regulation during bacterial infection. PUBLIC HEALTH RELEVANCE: Infectious bacteria sense and respond to various environmental cues. This project explores a novel signaling system in a plant-associated bacterium that coordinates external information with internal gene expression, in order to facilitate infection. Since this signaling system is also present in several animal pathogens, this project promises to yield broad molecular-level insights into host-microbe interactions.